Quantitative Shotgun Proteomics Associates Molecular-Level

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Quantitative shotgun proteomics associates molecular-level cadmium toxicity responses with compromised growth and reproduction in a marine copepod under multigenerational exposure Minghua Wang, Chen Zhang, and Jae-Seong Lee Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00149 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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Environmental Science & Technology

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-revised

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Quantitative shotgun proteomics associates molecular-level cadmium

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toxicity responses with compromised growth and reproduction in a

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marine copepod under multigenerational exposure

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Minghua Wang,*,†

Chen Zhang, †

Jae-Seong Lee*,†,‡

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†

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/College of the Environment & Ecology, Xiamen University, Xiamen, 361102, China

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‡

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Suwon 16419, South Korea

Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems

Department of Biological Science, College of Science, Sungkyunkwan University,

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ACS Paragon Plus Environment


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ABSTRACT: In this study, the copepod Tigriopus japonicus was exposed to different

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cadmium (Cd) treatments (0, 2.5, 5, 10 and 50 µg/L in seawater) for five generations

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(F0-F4), followed by a two-generation (F5-F6) recovery period in clean seawater. Six

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life-history traits (survival, developmental time of nauplius phase, developmental time

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to maturation, number of clutches, number of nauplii/clutch, and fecundity) were

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examined for each generation. Metal accumulation was also analyzed for generations

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F0-F6. Additionally, proteome profiling was performed for the control and 50 µg/L

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Cd-treated

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concentration-dependent manner, prolonging the development of the nauplius phase

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and maturation and reducing the number of nauplii/clutch and fecundity. However,

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during F5-F6, Cd accumulation decreased rapidly, and significant but subtle effects

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on growth and reproduction were observed only for the highest metal treatment at F5.

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Proteomic analysis revealed that Cd treatment had several toxic effects including

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depressed nutrient absorption, dysfunction in cellular redox homeostasis and

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metabolism, and oxidative stress, resulting in growth retardation and reproduction

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limitation in this copepod species. Taken together, our results demonstrate the

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relationship between molecular toxicity responses and population-level adverse

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outcomes in T. japonicus under multigenerational Cd exposure.

F4

copepods.

In

F0-F4

copepods,

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Cd

accumulated

in

a

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Table of Contents (TOC) Art

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Multigenerational exposure

Cadmium toxicity

Cellular redox homeostasis Nutrient absorption

49 Tigriopus japonicus

Metabolic disorders Oxidative stress

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Resisting

Producing toxic side-effects

Metabolic pathways Mitochondrial respiratory chain Oxidative phosphorylation

51

Physiological acclimation

Cadmium accumulation

Proteomic responses

Adverse outcomes

52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

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Cadmium (Cd) is a metal that is nonessential in most organisms (with the exception

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of marine diatom)1 and that can be toxic at even low doses. Cd toxicity has been

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attributed to the induction of oxidative stress,2 dysfunction in calcium homeostasis,3

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depletion of cellular sulfhydryl groups,4 and substitution for functional essential

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metals (e.g., zinc),4, 5 leading to cytotoxic events (e.g., lipid peroxidation and DNA

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damage). Cd thus produces multiple adverse effects in living organisms, including

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humans. In the planktonic marine copepod Centropages ponticus, 0.2 µg/L Cd

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exposure can cause oxidative stress by inducing lipid peroxidation, which

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significantly affects the enzyme activity and protein synthesis.6

INTRODUCTION

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Anthropogenic activities have led to Cd pollution becoming a severe environmental

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problem in many estuarine and coastal waters. In 2005 and 2006, water samples

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collected from 30 sites to explore the spatial distribution and temporal changes in

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dissolved metals in the seawater of Jinzhou Bay (China) revealed Cd concentrations

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from 1.65 to 2.01 µg/L.7 Indeed, Cd concentrations have reached 73.8 mg/L in some

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heavily polluted coastal areas (e.g., the Dardanelles).8 In such a seriously

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contaminated environment, the marine biota could be exposed to Cd pollution through

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many generations. Although many studies have examined the long-term effects of Cd

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pollution on freshwater organisms under multigenerational exposure,9-11 very little

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information has been presented concerning its multigenerational effects on the marine

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biota, particularly the mechanism behind these effects.

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The multigenerational effects of heavy metals (e.g., Cd, copper) have already been

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shown to include physiological acclimation and genetic adaption.9, 10,

12, 13

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context, physiological acclimation describes an individual's response that is rapidly

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observed under stress conditions and just as rapidly lost when the stress disappears.12

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Conversely, an adaptive response can involve changes in the genetic structure of a

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population (i.e., changes in allele frequency), which indicates that the adaptation

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potential could be maintained for a long period after the stressor is removed.14

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Nevertheless, the precise mechanisms of action for the multigenerational effects of Cd

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are still not fully understood.

In this

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Toxicoproteomics is a relatively new discipline that applies global proteomic

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technologies to toxicological studies to detect critical proteins/processes affected by

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environmental stressors.15 In previous studies, proteomic-based technologies have

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been applied to investigate the molecular response mechanisms of marine animals to

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heavy metal (e.g., Cd16 and mercury)17 pollution. In the copepod Tigriopus japonicus

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(a model species in marine stress ecology), tandem mass tag (TMT)-based

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quantitative proteomics was used to determine the molecular mechanism of mercury

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toxicity and tolerance under multigenerational exposure, and demonstrated that metal

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toxicity inhibits many crucial processes such as protein translation, macromolecule

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metabolism, DNA replication, the cell cycle, and vitellogenesis.17 However, the

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copepods are able to initiate several compensatory mechanisms (e.g., increased

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carbohydrate metabolism and enhancement of stress-related defense pathways) to

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defend against mercury attack.17

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In this study, the copepod T. japonicus was exposed to different concentrations of

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cadmium chloride (CdCl2) (nominal concentrations of 0, 2.5, 5, 10 and 50 µg/L in

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seawater) for five consecutive generations (F0-F4), followed by a recovery period of

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two generations (F5-F6) in seawater control conditions. Six life-history traits (survival

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rate, developmental time of nauplius phase, developmental time to maturation,

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number of clutches, number of nauplii/clutch, and fecundity) were examined for each

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generation. Cd accumulation analysis was also conducted in the adult copepods of

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each generation. Moreover, the proteome profiles of the F4 adult copepods were

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analyzed after multigenerational exposure to the control and 50 µg/L CdCl2 treatment.

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In this research, we sought to answer the following questions: Firstly, does

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physiological acclimation or genetic adaptation occur during multigenerational Cd

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exposure in marine copepods? Secondly, which proteins or protein functional groups

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are involved in that response? Lastly and, most importantly, what molecular

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mechanism drives the multigenerational effects of Cd in this copepod? Our study

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provides molecular insight into the physiological responses of individual marine

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copepods to Cd pollution under long-term multigenerational exposure.

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MATERIALS AND METHODS

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Copepod Maintenance. T. japonicus was originally collected in the rocky

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intertidal zone pools of Xiamen Bay, People's Republic of China (N 24°25.73'; E

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118°6.34') in 2007. The copepods were raised in our laboratory before the

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experiments and were maintained at a temperature of 22ºC in a 12/12 h light/dark

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cycle. Three algae (Isochrysis galbana, Platymonas subcordiformis, and Thalassiosira

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pseudonana) were equally mixed at a density of 5 × 107 cells/L, and the mixture was

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supplied as prey for the copepods. Seawater was obtained 20 km offshore in Xiamen

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Bay and filtered through a 0.22 µm polycarbonate membrane. The reference value for

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total Cd concentration in the seawater was 0.11 µg/L. The seawater characteristics

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were as follows: dissolved oxygen 6.2–6.7 mg/L; salinity 29–30 practical salinity

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units; and pH 8.0–8.1.

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Multigenerational Exposure. Based on the Chinese seawater quality standard (GB

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3097-1997), CdCl2 (Sigma-Aldrich, 99.5%) was added to four seawater samples to

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achieve nominal Cd2+ concentrations of 2.5, 5.0, 10, and 50 µg/L, and they, together

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with a seawater control without CdCl2, were used for the life-cycle tests of the

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copepods. Our 48-h acute testing revealed a median lethal concentration (LC50) for

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Cd exposure of 12.11 mg/L for T. japonicus (Figure S1), and the detailed protocols

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are presented in Text S1.

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Ten nauplii (< 24 h) per concentration treatment were transferred to 6-well tissue

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culture plates with 8 mL working volume in four replicates (i.e., a total of 40 nauplii).

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These nauplii were raised under the above-mentioned conditions until adult females

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produced egg sacs. The testing solutions were renewed daily (80% of the working

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volume). The algal culture was centrifuged to remove the culture medium (including

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nutrients and algal metabolites) and added at a density of approximately 5 × 107

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cells/L. We examined six life-history traits (i.e., survival rate, developmental period

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for nauplius phase, developmental time to maturation, number of clutches, number of

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nauplii per clutch, and fecundity) for each individual copepod in each generation.

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The survival rate (percentage) was calculated after exposure. Developmental stages

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were observed daily under a stereomicroscope and recorded to calculate the time of

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development from nauplii to copepodite (i.e., the nauplius phase) and from nauplii to

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adults with egg sacs (maturation). To measure the number of clutches, number of

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nauplii/clutch, and fecundity (offspring production), six females bearing an egg sac

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per replicate were individually transferred to a new 12-well plate with a 4 mL

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working volume. These females were reared under the above-mentioned conditions

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for 10 d. The resulting nauplii and unhatched clutches were counted under a

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stereomicroscope and removed.

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For the second generation (F1), 10 nauplii produced by F0 females (at the first or

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second brood) from each concentration treatment were transferred to new six-well

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plates. The experimental and exposure conditions were the same as those used for the

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F0 generation test. The copepods of subsequent generations were also treated with the

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same procedures as the F0 generation, and this multigenerational exposure was

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maintained until the F4 nauplii from the F3 generation developed to maturation. In

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addition, to determine Cd levels in the seawater solutions during multigenerational

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exposure, we conducted a 2-day exposure for all metal treatments, and the detailed

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procedure is provided in Text S2.

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Recovery. After that multigenerational exposure (F0-F4), the nauplii from both the

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F4 and F5 generations were placed in clean seawater until they developed to

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maturation (recovery for two generations). The experiments followed the same

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protocol used in the multigenerational exposure of F0-F4. Briefly, ten nauplii (< 24 h)

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per concentration treatment were cultured in six-well plates with four replicates. Also,

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the six life-history traits were observed for each individual copepod during the two

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recovery generations (F5-F6).

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Metal Accumulation Analysis. To measure Cd accumulation in copepods of each

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generation, we performed additional multigenerational exposure and recovery testing

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to secure the measurable quantity of Cd accumulation. The detailed procedures were

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the same as described above for F0-F6, except that 300 copepods were maintained in

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polycarbonate bottles with 400 mL of testing solution for this procedure, and each

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treatment was conducted in triplicate. The adult copepods were collected for each

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generation under the five treatments and subjected to Cd accumulation analysis, with

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three biological replicates per treatment. The copepods were washed with Milli-Q

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water three times to remove the Cd attached loosely to the carapace prior to metal

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analysis. After freeze-drying for 2 d, the copepods were digested in 70% nitric acid in

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a heating block at 80°C overnight. Cd accumulation in the digested samples was

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measured using inductively coupled plasma mass spectrometry (Agilent 7700x, USA).

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An internal standard (118In) was used to correct the instrumental drift. A quality

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control sample was measured after every ten samples, and the recovery percentage

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was 90–110%. Cd content in the copepods was measured as µg/g dry weight.

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Proteomic Analysis. To analyze the proteome profiles in the F4 T. japonicus

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copepods, a multigenerational exposure was simultaneously conducted. The copepods

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were exposed to the control and 50 µg/L Cd treatment for five generations (F0-F4).

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The detailed protocol was the same as that described above, but in this case, 100

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copepods were raised in polycarbonate bottles with 200 mL testing solution, and each

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treatment was repeated with four replicates. After exposure, two replicates

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(approximately 200 copepods) were pooled for each treatment and immediately stored

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at -80°C for follow-up proteomic analysis (two experimental replicates per treatment).

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Protein Labeling and Strong Cation Exchange (SCE) Fractionation. Proteins were

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extracted, quantified and labeled using the TMT-6plex Kit (ThermoFisher Scientific,

205

USA) following the manufacturer's instructions. Protein samples were labeled with

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TMT tags using the following protocol: tags 126 and 127 were designated to label the

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two biological replicates for the F4 control group, with 128 and 129 used for the two

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replicates in the F4 treated group. All four samples were pooled and dried with

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vacuum centrifugation. The peptide mixtures were fractionated using SCE

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chromatography, followed by liquid chromatography-tandem mass spectrometry

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(LC-MS/MS) analysis. The detailed procedures are provided in Text S3.

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LC-MS/MS Analysis and Database Searches. The peptides were analyzed using a Q

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ExactiveTM Plus hybrid quadrupole-Orbitrap mass spectrometer coupled with an

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EASY-nLC 1000 UPLC system (ThermoFisher Scientific, USA). The obtained

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peptide sequences were searched against the NCBI_Tigriopus proteome (1659

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sequences) and the T. japonicus transcriptome (a total of 46369 sequences from a

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previous work18 and our published data)19 database. The detailed protocols are

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provided in Text S4.

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Bioinformatic Analysis. Differentially expressed proteins (DEPs) were identified

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only if the normalized fold change was higher than 1.30 (up-regulated) or lower than

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0.77 (down-regulated), which was calculated as the 95% confidence level based on

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pairwise analysis between two experimental replicates.20 DEPs were annotated into

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three categories based on Gene Ontology (GO) terms: biological process, cellular

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component, and molecular function. The protein domain function annotation was

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defined with InterProScan using the protein sequence alignment method

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(http://www.ebi.ac.uk/interpro/), and the protein pathway was annotated using the

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Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Enrichment analysis

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was conducted for GO terms, protein domain and KEGG pathway using the Database

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for Annotation, Visualization, and Integrated Discovery. Statistically significant

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enrichments were identified using Fisher's exact test with a Benjamini-Hochberg's

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corrected p value of < 0.05.

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Biochemical Parameter Determination. To verify our proteomic analysis, a

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multigenerational exposure experiment was repeated independently; that is, the

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copepods were exposed to the control and 50 µg/L Cd treatment for five generations

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(F0-F4). The detailed procedure was the same as that were previously described for

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proteomic analysis. After exposure, several biochemical parameters were specifically

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measured in the F4 copepods. Namely, the enzymatic activities of chymotrypsin-like

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proteinase, alcohol dehydrogenase, cytochrome c oxidase, and glutathione peroxidase,

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as well as the level of lipid peroxidation (LPO) were detected using the

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manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

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The detailed protocols are available in Text S5.

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Statistical Analysis. All experiments were repeated at least three times (n = 3–4),

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and all the data are presented as mean ± standard deviation. Statistical analysis was

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performed using SPSS 19.0 software. One-way ANOVA and Fisher least significant

245

difference test were used to evaluate whether the means differed significantly among

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the groups. Differences were considered significant at p < 0.05. Prior to one-way

247

ANOVA, data were log transformed to meet ANOVA assumptions of normality and

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variance homoscedasticity.

249 250

RESULTS

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Exposure Verification. Measured concentrations of total Cd in seawater were

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comparable to nominal concentrations for all metal treatments during a 2-day

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exposure. On average, differences were < 15% (Table S1). In each case, more than 95%

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of total Cd existed in the dissolved phase in the tested seawater, suggesting that the

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nominal concentrations reflected the real exposure conditions under which the

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copepods were maintained during multigenerational exposure.

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Metal Accumulation. For F0-F4, Cd accumulated significantly (p < 0.05) in the

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adult copepods in a dose-dependent manner in each generation (Table 1). At the same

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metal exposure level, Cd accumulation tended to increase with generations from F0 to

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F4. For example, the copepod Cd content in the control was less than 0.13 µg/g for

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F0-F4. However, the Cd content in the 50 µg/L Cd treatment was 71.43, 78.19, 80.03,

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90.58 and 109.94 µg/g Cd for F0-F4, respectively. Interestingly, when all the

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copepods were placed in clean seawater for two generations (F5-F6), metal

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accumulation in the exposed animals decreased rapidly. In F5, Cd content in the

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copepods pre-exposed to the highest Cd treatment (50 µg/L) was 0.52 µg/g,

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significantly higher than the 0.07 µg/g content in the control. However, in generation

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F6, the Cd content in the copepods did not vary between treatment groups.

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Multigenerational Effects. To test the effects of five successive generations of

269

exposure to waterborne Cd, we examined six life-history traits of copepod T.

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japonicus under a multiple life-cycle test (Figure 1). The survival rate did not vary

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significantly between treatments in any of the generations. Similarly, Cd treatment did

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not affect the number of clutches for the copepods during multigenerational exposure,

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except that the number of clutches decreased (p < 0.05) in F4 with metal treatments of

274

5, 10, and 50 µg/L.

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Nauplius phase and development time were significantly delayed (p < 0.05) by Cd

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treatment in most cases, and the impacts on both traits displayed a dose-dependent

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response in later generations. For instance, development time from nauplius to adult

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under the control condition was 13.3, 13.4, 13.3, 13.3 and 13.1 d for F0-F4, but was

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increased to 14.2, 14.8, 15.1, 15.2 and 15.3 d for F0-F4 in the 50 µg/L Cd exposure

280

group (Figure 1C). Likewise, Cd exposure significantly inhibited (p < 0.05) the

281

number of nauplii per clutch and fecundity under most circumstances, and the

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inhibitory effects tended to be dose-dependent. For example, total fecundity under the

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control condition was 100.4, 96.8, 103.4, 101.1 and 101.4 for F0-F4 but decreased to

284

85.8, 81.6, 79.2, 78.1 and 77.9 under the 50 µg/L Cd treatment (Figure 1F). Thus, the

285

effects of Cd exposure on the nauplius phase, development time, number of

286

nauplii/clutch, and total fecundity tended to become more severe over the generations.

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Recovery. When all of the copepods were recovered in clean seawater for two

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generations, the six traits were not significantly different among treatments, except

289

that the highest 50 µg/L Cd treatment had negative impacts on the F5 generation

290

(Figure 2). The highest CdCl2 treatment significantly lengthened (p < 0.05) the

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developmental times for nauplius phase and maturation and also significantly reduced

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(p < 0.05) the number of nauplii/clutch and total fecundity of the F5 copepods.

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Proteome Profiles. Analysis with LC-MS/MS produced 24991 peptide spectra,

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which resulted in 2626 identified proteins, of which 1672 were quantified. In total,

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there were 58 DEPs caused by Cd treatment, with 28 up-regulated proteins and 30

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down-regulated ones (Tables S2 and S3). Reproducibility was analyzed for the two

297

experimental replicates based on DEPs ratios with a regression coefficient greater

298

than 0.90 (Figure S2). Meanwhile, DEPs were classified into different groups:

299

metabolic process, single-organism process, cellular process, localization, biological

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regulation, response to stimulus, and other related functions (Figure S3). Most DEPs

301

were involved in metabolic processes, single-organism processes, and cellular

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processes. Additionally, DEPs were found in several cellular compartments:

303

extracellular, cytosol, mitochondrion, nuclear, and plasma membrane (Figure S3).

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To investigate functional differences in up-regulated and down-regulated proteins,

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the quantified proteins were analyzed via GO terms, protein domain and KEGG

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pathway enrichment-based analysis (Figures 3–5; detailed information is provided in

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Tables S4 and S5). With regard to up-regulated proteins, many significantly enriched

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important processes were classified as cellular component, molecular function, KEGG

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pathway, and protein domain proteins. Cellular component analysis revealed that

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several categories including mitochondrial inner membrane (GO:0005743),

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mitochondrion

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mitochondrial respiratory chain (GO:0005746) were increased by Cd treatment. In the

313

molecular function category, catalytic activity (GO:0003824), cytochrome-c oxidase

314

activity (GO:0004129), electron carrier activity (GO:0009055), transmembrane

315

transporter activity (GO:0022857), and others were enhanced by Cd exposure. KEGG

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pathway analysis demonstrated that most up-regulated pathways were enriched into

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metabolic pathways (ko01100), oxidative phosphorylation (ko00190), and several

318

diseases, e.g., Alzheimer's disease (ko05010), non-alcoholic fatty liver disease

319

(ko04932), and Parkinson's disease (ko05012). Additionally, protein domains (the

320

nucleoside

321

molybdopterin-binding domain [IPR000572], and ribosomal protein L11, N-terminal

322

[IPR020784]) were up-regulated under Cd treatment.

(GO:0005739),

phosphorylase

cytochrome

domain

complex

(GO:0070069),

[IPR000845],

and

oxidoreductase,

323

In the case of down-regulated proteins, significant enrichments were classified

324

based on the KEGG pathway and protein domain categories. The main KEGG

325

pathways of neuroactive ligand-receptor interaction (ko04080) and protein digestion

326

and absorption (ko04974) were depressed by Cd exposure. Protein domains such as

327

alcohol

328

(IPR016040), serine proteases, and trypsin domain (IPR001254) were inhibited by Cd

329

exposure.

330

dehydrogenase,

C-terminal

(IPR013149),

NAD(P)-binding

domain

Biological Parameters. Our proteomic data were supported by a biochemical assay

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331

for several enzyme activities in the F4 copepods (Figures S4 and S5); that is to say,

332

the enzyme activities from the biochemical assay were consistent with the proteomic

333

data in terms of both changes in direction and magnitude. Taking chymotrypsin-like

334

proteinase as an example, the activity and expression were decreased by 0.43 and 0.67

335

times, respectively, under Cd exposure (Figure S4). Additionally, in contrast to the

336

control, Cd treatment significantly enhanced the LPO level (4.05 fold) in the exposed

337

copepods (Figure S6), partially lending a support to the notion that Cd could result in

338

several toxic events including oxidative stress and consequently produced

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population-level responses (Figure 6).

340 341

342

Although many studies have investigated the effects of Cd on growth and

343

reproduction in aquatic invertebrates under multigenerational exposure scenarios,9-11

344

ours is significant in its attempt to examine the impacts of multigenerational exposure

345

to Cd on a wide range of traits in a marine copepod, T. japonicus. We found that metal

346

accumulation in the treated copepods over generations negatively affected growth and

347

reproduction in a concentration-dependent manner. We also investigated copepod

348

proteomes after multigenerational Cd exposure and found that phenotypic plasticity

349

could partially account for its tolerance against Cd toxicity. In particular, using GO

350

terms, protein domain, and KEGG pathway-based enrichment analyses, we were able

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to determine the functional roles of several critical proteins/processes, which provided

352

a mechanistic explanation for both Cd toxicity and the Cd tolerance of this marine

DISCUSSION

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copepod. We thus combined comparative proteome profiling with physiological

354

observations (e.g., developmental time and reproductive performance) in a marine

355

copepod, linking the copepod's proteome changes under multigenerational Cd

356

exposure with the consequent adverse outcomes at a population level.

357

Multigenerational Effects. Our study revealed that during multigenerational

358

exposure, Cd significantly accumulated (p < 0.05) in the treated animals in a

359

dose-dependent manner. Dry-weight concentration factors (DCFs) of Cd in the

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exposed copepods were 721–1556, 1418–1564, 1464–1732, 1812–2216 and

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2157–3544 L/kg for F0, F1, F2, F3 and F4, respectively, which are comparable with

362

the DCFs of other marine copepods.21, 22 Meanwhile, the treated Cd levels in our work

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were within the range of Cd concentrations found in several marine copepods in field

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studies,23, 24 supporting the environmental relevance of our findings. Moreover, at the

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same metal exposure level, Cd accumulation tended to increase over the generations

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from F0 to F4. This tendency of Cd to accumulate over generations could be

367

attributed to maternal transfer of the metal in the treated animals during

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multigenerational exposure, as shown in previous studies.10,

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concentration-dependent Cd accumulation in the first generation of recovery (F5)

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supports the maternal metal transfer theory because all copepods were returned to

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clean seawater for the F5 generation. Taken together, Cd accumulation caused

372

significant proteome changes, and the consequent adverse outcomes at the population

373

level included extended developmental time and reduced reproductive capacity in the

374

exposed copepods.

17


25

Actually, the


375

Our study indicates that Cd exposure does not significantly affect the survival rate

376

and number of clutches under most circumstances during multigenerational exposure

377

in generations F0-F4, but it does delay (p < 0.05) development to the nauplius phase

378

and maturation and significantly limit (p < 0.05) the number of nauplii per clutch and

379

total fecundity of these copepods. Furthermore, those negative effects were

380

dose-dependent for each generation, which is mainly attributable to increased Cd

381

accumulation. To the best of our knowledge, no previous work has concentrated on

382

multigenerational Cd toxicity in marine invertebrates. The few studies conducted on

383

Cd toxicity in marine copepods have focused on toxic effects after exposure in no

384

more than one generation.26, 27 For example, a recent early life stage toxicity test

385

indicated that 6.87–110 µg/L of Cd exposure dramatically decreases the larval

386

development ratio and hatching success in the marine copepod Acartia tonsa in a

387

concentration-dependent manner,27 which is consistent with our study. Collectively,

388

the inhibitory impacts of Cd on growth and reproduction are likely due to energy

389

redistribution (i.e., energetic trade-off) in copepods under metal exposure, which has

390

also been observed in several studies.10, 17, 28, 29 The treated copepods might have

391

increased energetic allocation to defense/repair processes (metal tolerance) during Cd

392

exposure and consequently displayed inferior growth and reproduction. It is worth

393

noting that Cd accumulation in the treated copepods could also have directly

394

disrupted their physiological integrity and have inhibited overall fitness including

395

growth and reproduction, as an individual's physiological performance can ultimately

396

be affected by any stressors present at a sufficiently high level or for a long enough

18


Page 18 of 42

Page 19 of 42


397

period of time.30

398

Recovery. After exposure to Cd for five generations, all copepods were transferred

399

into clean seawater for two generations (F5-F6) as a recovery period. Cd

400

accumulation in the F5 copepods was slightly but significantly increased (p < 0.05) by

401

pre-exposure to Cd concentrations of 5–50 µg/L, although significant effects on

402

developmental time and reproductive performance in this generation were found only

403

in the highest Cd exposure group (50 µg/L). Moreover, no significant Cd

404

accumulation was observed in the copepods during the second recovery generation

405

(F6), and all the measured life-history traits also recovered to control levels. Thus,

406

maternal transfer of Cd occurred in the F5 copepods, which exerted a subtle

407

carry-over effect on this generation. A full or partial recovery within one copepod

408

generation excludes the effects of rapid adaptation to Cd exposure as a possible cause;

409

therefore, physiological acclimation probably played a role in the multigenerational

410

effects of Cd in this study, which is in accordance with several previous studies

411

examining the multigenerational effects of copper12 or mercury25 on marine copepods.

412

For example, our previous study25 revealed that mercury toxicity significantly affects

413

growth and reproduction in T. japonicus under multigenerational exposure, but that

414

the negative effects disappear completely after a single generation, indicating that

415

physiological acclimation (i.e., phenotypic plasticity) is primarily responsible for the

416

multigenerational effects of mercury in this copepod. The present study together with

417

our previous one25 suggests that the harmful effects of metals (e.g., Cd and mercury)

418

on T. japonicus at the individual or population level could resolve as soon as metal

19



419

pollution is eliminated from the environment due to the phenotypic plasticity

420

displayed by this copepod.

421

Proteomes Altered by Cd Exposure Account for Population Response. As

422

discussed above, multigenerational exposure to environmentally relevant Cd

423

concentrations dramatically increased Cd accumulation, leading to negative impacts

424

on growth and reproduction in the copepod T. japonicus, which was reflected in our

425

proteomic results. In brief, Cd exposure inhibited several critical processes including

426

protein digestion and absorption and cellular redox homeostasis, despite

427

compensatory mechanisms that enhanced cellular energy production in the defense

428

against metal toxicity. Specifically, depressed protein digestion and absorption likely

429

indicated decreased feeding activity with a concurrent decrease in total energy input

430

into the treated copepods. Meanwhile, increased energy production in the exposed

431

animals caused some toxic side-effects, namely oxidative stress (Figure S6) and

432

metabolism deregulation. Overall, the significantly altered proteomes led to

433

dysfunction in the physiological integrity of the Cd-treated copepods that ultimately

434

translated into adverse outcomes at the population level, i.e., growth retardation and

435

limited reproduction (Figure 6).

436

One important KEGG pathway of protein digestion and absorption (three proteins)

437

was depressed by Cd exposure. These three proteins were also enriched in protein

438

degradation (proteolysis). In copepods, protein digestion and absorption involves a

439

series of digestive enzymes (e.g., carboxypeptidase B and chymotrypsin-like

440

proteinase in our study) to break down ingested proteins into components that can

20


Page 20 of 42

Page 21 of 42


441

easily be absorbed and directed into the cellular metabolism. In addition, two protein

442

domains, namely serine proteases, trypsin domain (three proteins) and peptidase S1,

443

PA clan (three proteins) were down-regulated by Cd treatment, providing supportive

444

evidence for inhibitory proteolysis in the treated copepods. Decreased protein

445

assimilation and proteolysis likely reflect reduced digestion in the affected copepods,

446

probably resulting from depressed feeding performance, especially considering that

447

excess food was provided during exposure. Four down-regulated proteins,

448

alpha-amylase (fragment), cellobiohydrolase CHBI, endo-beta-1, 4-mannanase, and

449

putative endo-1, 3(4)-beta-glucanase, could be responsible for hydrolyzing dietary

450

saccharides in this copepod, but they were not significantly enriched, suggesting that

451

nutrient adsorption was depressed by Cd exposure. Our proteomic study suggested

452

that metal exposure might depress the treated copepods' feeding activity and

453

subsequently decrease their total energetic input, consequently reducing the energy

454

allocation available for growth and reproduction. Moreover, the consequently

455

depressed energetic input might affect the energetic trade-off between Cd tolerance

456

and growth/reproduction, particularly in cases where Cd treatment inhibits copepod

457

digestive processes, as shown in Cd-exposed daphnids.31, 32

458

Two protein domains, alcohol dehydrogenase, C-terminal and GroES (chaperonin

459

10)-like, were decreased by Cd treatment. GroES (chaperonin 10) acts as a molecular

460

chaperone, which functions in protein folding and possibly intercellular signaling.

461

Those two processes were enriched by four overlapping proteins: two proteins

462

referring to the alcohol dehydrogenase GroES domain protein, the alcohol

21



463

dehydrogenase

464

12-hydroxydehydrogenase. The alcohol dehydrogenases include a group of isozymes

465

that can catalyze the oxidation of primary and secondary alcohols to aldehydes and

466

ketones by reducing nicotinamide adenine dinucleotide (NAD+) to NADH,33 and they

467

thus

468

12-hydroxydehydrogenase catalyzes leukotriene B4 to the much less active metabolite

469

12-oxo-leukotriene B4, inhibiting the pro-inflammatory actions of leukotriene B4,

470

which has been implicated in several allergic and inflammatory diseases in humans.34

471

Notably, all four proteins display zinc ion binding and are also involved in

472

oxidation-reduction processes. Our proteomic analysis thus demonstrated that Cd

473

toxicity compromised cellular redox homeostasis in the exposed copepods by

474

disrupting several crucial proteins/enzymes via substitution for functionally essential

475

zinc.

play

an

zinc-binding

important

role

domain

in

protein,

ethanol

and

Page 22 of 42

metabolism.

leukotriene

Leukotriene

B4

B4

476

Interestingly, many enriched processes consisted of proteins up-regulated under Cd

477

exposure, including mitochondrion, cytochrome complex, mitochondrial respiratory

478

chain, organelle inner membrane, catalytic activity, cytochrome-c oxidase activity,

479

electron carrier activity, metabolic pathways, and oxidative phosphorylation.

480

Generally speaking, most of the up-regulated processes were related to energy

481

metabolism, which might have improved the treated copepods' resistance to Cd

482

toxicity. Some of the proteins involved in energy production (e.g., mitochondrial

483

respiratory chain, mitochondrion and oxidative phosphorylation) are GRIM-19,

484

cytochrome b-c1 complex subunit 7, AGAP000851-PA, cytochrome c oxidase subunit

22


Page 23 of 42


485

Va, and vacuolar ATP synthase subunit B. GRIM-19 is a cell death regulatory protein

486

that plays an essential role in the assembly and function of mitochondrial complex I.35

487

Similarly, cytochrome b-c1 complex subunit 7, AGAP000851-PA, and cytochrome c

488

oxidase subunit Va display cytochrome oxidase activity and are involved in

489

mitochondrial electron transfer in cells, ultimately facilitating energy production. The

490

increased expression of those proteins suggests an enhanced energy yield in the

491

exposed copepods as a compensatory reaction to Cd toxicity. It is important to note

492

that the metabolic pathways (16 proteins) contained triosephosphate isomerase and

493

fructose-1,6-bisphosphase. Triosephosphate isomerase catalyzes the reversible

494

interconversion of the triose phosphate isomers (dihydroxyacetone phosphate and

495

glyceradehyde

496

glycolysis/gluconeogenesis. Fructose-1,6-bisphosphase, which is a rate-limiting

497

enzyme,

498

gluconeogenesis.37 The up-regulated expression of triosephosphate isomerase and

499

fructose-1,6-bisphosphase thus leads to enhanced gluconeogenesis and, as a

500

consequence, glucose accumulation in metal-exposed animals. This could probably be

501

regarded as a compensatory response to the low absorption of dietary saccharides

502

caused by decreased expression of several saccharide hydrolyzing enzymes

503

(alpha-amylase, cellobiohydrolase CHBI, endo-beta-1, 4-mannanase, and putative

504

endo-1, 3(4)-beta-glucanase in this study) under Cd exposure. In addition, five

505

up-regulated

506

AGAP000851-PA, cytochrome c oxidase subunit Va, and insulinase) are enriched in

3-phosphate),36

converts

and

is

fructose-1,6-bisphosphate

proteins

(GRIM-19,

cytochrome

therefore

to

fructose

b-c1

23


involved

6-phosphate

complex

subunit

in

in

7,


507

several human diseases including Alzheimer's disease, non-alcoholic fatty liver

508

disease, and Parkinson's disease, suggesting that metabolic dysfunction could also

509

lead to adverse health in treated copepods. Such neurodegenerative disorders might

510

not be observed in copepods; however, this connection could help in explaining

511

metabolic dysregulation in the treated animals. Taken together, the present proteomic

512

work demonstrated that Cd exposure disrupted the energy metabolism of the treated

513

copepods and is thus likely to be associated with the induction of several metabolic

514

disorders, despite compensatory energy production mechanisms in the exposed

515

copepods that help counteract metal toxicity.

516

Although increased energy production via oxidative phosphorylation allowed the

517

copepods to defend against Cd toxicity, reactive oxygen species may be produced,

518

ultimately resulting in cellular oxidative stress, an idea supported by our proteomic

519

analysis. Two proteins correlated with oxidative stress were altered by Cd treatment.

520

Glutathione reductase catalyzes the reduction of glutathione disulfide (GSSG) to the

521

sulfhydryl form glutathione (GSH), which is a critical antioxidant preventing

522

oxidative stress and maintaining the reducing environment of cells.38 Glutathione

523

peroxidase is responsible for reducing lipid hydroperoxides to their corresponding

524

alcohols and reducing free hydrogen peroxide to water, with GSH being concurrently

525

oxidized into GSSG, thus protecting the organisms from oxidative damage. Increased

526

expression of glutathione reductase, together with a down-regulation of glutathione

527

peroxidase, enhances GSH levels and maintains the cellular reducing environment.

528

This probably acts as a compensatory response against the oxidative stress caused by

24


Page 24 of 42

Page 25 of 42


529

Cd toxicity. The above hypothesis was, to some extent, confirmed by the increased

530

expression of two proteins (chaperone protein dnaJ15 and probable protein

531

disulfide-isomerase A6) involved in stress responses. In summary, protein alterations

532

due to stress suggested that, although the compensatory mechanisms related to energy

533

production were initiated by the treated copepods to defend against Cd toxicity, the

534

concurrent side-effects, including oxidative stress (Figure S6) and metabolic

535

dysfunction, affected physiological integrity and ultimately compromised growth and

536

reproduction of exposed copepods.

537

Implications. Our proteomic study indicates that Cd multigenerational toxicity

538

resulted in a range of toxic effects (e.g., depressed nutrient absorption, dysfunction in

539

cellular redox homeostasis and metabolism, and oxidative stress), despite increases in

540

energy production as a compensatory reaction to metal exposure. In particular, the

541

probable energetic trade-offs between Cd tolerance and growth/reproduction were

542

likely affected by decreased total energetic input due to poor nutrient absorption under

543

Cd exposure. Molecular-level perturbations are frequently critical toxic events

544

predicting adverse outcomes at higher levels of biological organization.39 One

545

conceptual framework, the adverse outcome pathway (AOP), has gained increasing

546

acceptance in the context of regulatory ecotoxicology.40 An AOP is capable of

547

establishing the biologically causal linkages between molecular toxicity effects and

548

higher-level adverse outcomes in biological organization, such as growth and

549

reproduction. Toxic events ultimately produce adverse outcomes at higher levels, in

550

this case of growth delay and reproduction limitation in the exposed copepods, thus

25



551

linking molecular toxicity responses with population-level effects (Figure 6). Our

552

ecotoxicoproteomic analysis could not provide information on epigenetic changes

553

(e.g., DNA methylation), however, which may have an important role in Cd

554

multigenerational effects on marine copepods; thus, further investigation is needed.

555

Nevertheless, the outcomes of this proteomic study contribute to our understanding of

556

how molecular changes translate into population-level responses in the presence of

557

heavy metal pollution in marine environments, especially coastal waters. Recalling

558

that food was provided ad libitum during multigenerational exposure in this study, the

559

negative effects of Cd on growth and reproduction of marine copepods could be

560

aggravated under non-ideal food conditions, e.g., the limited quantities likely to be

561

encountered in future oceans. Correspondingly, further study is required to examine

562

how food conditions, especially food limitation, will affect individual marine copepod

563

responses to heavy metal pollution, including Cd exposure.

564 565

AUTHOR INFORMATION

566

Corresponding Author

567

*Phone: +86-592-2880219; fax: +86-592-2880219;

568

e-mail: [email protected]; [email protected]

569 570

Notes

571

The authors have no competing financial interests to declare.

572

26


Page 26 of 42

Page 27 of 42


573

574

This work was supported by the National Natural Science Foundation of China (no.

575

41476094) and the Natural Science Foundation of Fujian Province of China (no.

576

2017J01081). We would like to thank Professor John Hodgkiss of the City University

577

of Hong Kong for his assistance with English in this manuscript.

ACKNOWLEDGEMENTS

578 579

580

Additional details for acute toxicity testing, a 2-day exposure, proteomic analysis, and

581

biochemical parameter determination, as well as related data are available in the

582

Supporting Information.

ASSOCIATED CONTENT

583 584

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585

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33



Page 34 of 42

713

Table 1. Metal accumulation in the copepod Tigriopus japonicus under multigenerational exposure to different cadmium treatments (control, 2.5,

714

5, 10, and 50 µg/L). Data are described as mean ± standard deviation (n = 4). Different letters indicate a significant difference among different

715

cadmium treatments at p < 0.05.

716

Treatments

Cadmium accumulation (µg/g)

(µg/L)

F0

F1

F2

F3

F4

F5 (recovery)

F6 (recovery)

control

0.04± 0.00a

0.10±0.01a

0.05±0.01a

0.18±0.06a

0.13±0.01a

0.07±0.00a

0.06±0.01a

2.5

3.89±0.58b

3.90±0.22b

4.33±0.23b

5.54±0.17b

8.86±0.44b

0.07±0.00a

0.06±0.02a

5

5.14±0.43b

7.09±0.90c

7.32±0.06c

10.32±0.04c

10.80±1.08c

0.14±0.02b

0.05±0.01a

10

7.21±0.51c

15.36±0.34d

16.02±0.39d

21.38±1.68d

21.57±0.37d

0.16±0.01b

0.05±0.01a

50

71.43±2.01d

78.19±1.95e

80.03±0.60e

90.58±0.41e

109.94±0.17e

0.52±0.06c

0.05±0.01a

717 718 719

34


Page 35 of 42


720

Figure Captions

721

Figure 1. Survival rate (A), nauplius phase (B), development time (C), number of

722

clutches (D), number of nauplii per clutch (E), and fecundity (F) in five generations of

723

Tigriopus japonicus exposed to different cadmium treatments (control, 2.5, 5, 10, and

724

50 µg/L). Data are described as mean ± standard deviation (n = 4). Different letters

725

indicate a significant difference among different metal treatments at p < 0.05.

726

Figure 2. Survival rate (A), nauplius phase (B), development time (C), number of

727

clutches (D), number of nauplii per clutch (E), and fecundity (F) in the copepod

728

Tigriopus japonicus transferred into clean conditions after five generations of

729

exposure to different cadmium treatments (control, 2.5, 5, 10, and 50 µg/L). F5

730

represents the first generation during recovery, and F6 for the second generation. Data

731

are described as mean ± standard deviation (n = 4). Different letters indicate a

732

significant difference among different treatments at p < 0.05.

733

Figure 3. GO terms-based enrichment analysis was performed for differentially

734

expressed proteins caused by cadmium exposure. Differentially expressed proteins

735

were divided into two groups (up-regulated and down-regulated). It should be noted

736

that only the up-regulated proteins were significantly enriched in the two categories,

737

namely cellular component and molecular function. Significant enrichments were

738

calculated at a corrected p value of < 0.05.

739

Figure 4. KEGG pathway-based enrichment analysis was performed for differentially

740


741

were divided into two groups (up-regulated and down-regulated). Significant

35



742

enrichments were calculated at a corrected p value of < 0.05.

743

Figure 5. Protein domain-based enrichment analysis was performed for differentially

744


745

were divided into two groups (up-regulated and down-regulated). Significant

746

enrichments were calculated at a corrected p value of < 0.05.

747

Figure 6. Proteomics provides a putative mechanism into the physiological

748

acclimation used by Tigriopus japonicus to fight against cadmium multigenerational

749

toxicity under long-term exposure. Cadmium toxicity inhibited several important

750

processes including cellular redox homeostasis and nutrient absorption. In particular,

751

depressed nutrient absorption caused a decreased total energy input in the treated

752

copepods. To resist Cd toxicity, the copepods might have initiated some compensatory

753

systems, especially concerning increased cellular energy production. However, the

754

enhanced energy production also resulted in some toxic side-effects, e.g., metabolism

755

dysfunction and oxidative stress. Collectively, the toxic events produced adverse

756

outcome pathways at the population level; thus the copepods' growth and

757

reproduction were compromised by multigenerational cadmium exposure. Note: the

758

red arrow indicates the processes down-regulated by cadmium toxicity, and the blue

759

arrow for the up-regulated processes initiated by physiological acclimation.

760 761 762 763

36


Page 36 of 42

Page 37 of 42


Figure 1

764 765 110

A

a a

100

a a

a

a

a

a a a a

a

a

a a a

a

a a a a

a

B

6.5 6.0

a

a a

5.5

90

a

a a

a a

a ac

bc b b

d c c

b

b b b

c

c

b b b a

a

a

5.0

Nauplius phase (d)

Survival rate (%)

80 70 60 50 40 30

4.5 4.0 3.5 3.0 2.5 2.0 1.5

20

1.0

10

0.5

0

0.0

C

F3

D b

b

15 14

ab ab ab

a a a

a

b b

a

bc

c

bc

b b

c b

a

a

c

F1

F0

F4

17 16

F2

4.0

F3

F4

766

c c

a

3.5

a

a a a a

a a

a

13

3.0

12

Number of clutch

Development time (d)

F2

F1

F0

11 10 9 8 7 6 5

a a

a

a a a a

a

a a a a

a

b

ac

bc bc

2.5 2.0 1.5 1.0

4 3

0.5

2 1

0.0

0

F1

F0

E

F2

F3

F0

F4

F2

F4

F3

40

F

a a

35

ab

a

ab bc

ab

a b

b

b

30

a b

b b b

b b c

b

b

120

ab bc

a a

ab

ad

bcd

a

a

ab b

100

bc c

a b

b b

b cd

b b b b

b

b

b b b

80

25

Fecundity/10 d

Number of nauplii/clutch

F1

20 15

60

40

10 20

5 0

0

F0

F1

F2

F3

F0

F4

F1

control

F3

Generation

Generation

767

F2

2.5 µg/L

5 µg/L

768 769

37


10 µg/L

50 µg/L

F4

b


Page 38 of 42

Figure 2

770 771

B

110

A

a 100

a

a

a

a

a

a

a

a

a

6.5 6.0 5.5

90

b a

a

Nauplius phase (d)

5.0

Survival rate (%)

80 70 60 50 40

a

a

a

a

a

a

a

a

a

4.5 4.0 3.5 3.0 2.5 2.0

30 1.5

20

1.0

10

0.5 0.0

0

F5

Development time (d)

15 14 13

F5

D

17 16

ab a

ab ab

a

a

a

a

a

12 11 10 9 8 7 6 5

a

a

a

a

a

a

2.5 2.0 1.5

0.5

1 0

0.0

F5

F6

40

a a

a

a

a

a

a

F5 a

F

F6

120

a

a

a 100

b

b

a

a

ab ab c

30

Fecundity/10 d

Number of nauplii/clutch

a

1.0

2

35

F6

772

a

3.0

4 3

E

3.5

b

Number of clutch

C

F6

25 20 15

80

60

40 10

20 5

0

0

F5

F5

F6

773

control

F6

Generation

Generation

2.5 µg/L

5 µg/L

10 µg/L

774 775

38


50 µg/L

a

a

Page 39 of 42


776

Figure 3

777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797

39



798

Figure 4

799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819

40


Page 40 of 42

Page 41 of 42


820

Figure 5

821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841

41



842

Figure 6

843 844 845

Cadmium toxicity Cellular redox homeostasis Nutrient absorption

846 847

Metabolic disorders Oxidative stress Resisting

848 849

Producing toxic side-effects

Adverse outcome pathways at a population level, e.g., growth delays and reproduction limitations

Metabolic pathways Mitochondrial respiratory chain Oxidative phosphorylation

850

Physiological acclimation 851 852 853

42


Page 42 of 42

Quantitative Shotgun Proteomics Associates Molecular-Level

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